Collagen is the major structural constituent of mammalian organisms and makes up a large portion of the total protein content of skin and other parts of the animal body. In humans, it is particularly important in the wound healing process and in the process of natural aging. Various skin traumas such as burns, surgery, infection and accident are often characterized by the erratic accumulation of fibrous tissue rich in collagen and having increased proteoglycan content. In addition to the replacement of the normal tissue which has been damaged or destroyed, excessive and disfiguring deposits of new tissue sometimes form during the healing process. The excess collagen deposition has been attributed to a disturbance in the balance between collagen synthesis and collagen degradation.
Numerous diseases and conditions are associated with excess collagen deposition and the erratic accumulation of fibrous tissue rich in collagen. Such diseases and conditions are collectively referred to herein as “collagen-mediated diseases”. Collagenase, an enzyme that has the specific ability to digest collagen, has been used to treat a variety of collagen-mediated diseases. Collagenase formulations have a broad variety of uses in the medical field, including their use as therapeutics.
Collagenase for use in therapy may be obtained from a variety of sources including mammalian (e.g. human), crustacean (e.g. crab, shrimp), fungal, and bacterial (e.g., from the fermentation of Clostridium, Streptomyces, Pseudomonas, or Vibrio). Collagenase has also been genetically engineered. One common source of crude collagenase is from a bacterial fermentation process, specifically the fermentation of Clostridium histolyticum (“C. histolyticum”). The crude collagenase obtained from C. histolyticum may be purified using any of a number of chromatographic techniques.
Collagenases from C. histolyticum, i.e., collagenase class I and class II, are of particular importance, e.g., for dissociation of organ tissue in vitro. Importantly, collagenase digestion of pancreatic tissue is presently used in the preparation and isolation of human islet cells. However, a number of other different specific cell types have been isolated from attendant connective tissue, including fat cells from adipose tissue, hepatocytes from liver, chondrocytes from cartilage, myocytes from heart, and osteoblasts from bone. (Ian Freshney, Culture of animal cells, A Manual of Basic Technique, Alan R Liss Inc., 1987.)
A practical advantage of using C. histolyticum for the production of collagenases is that it can be cultured in large quantities in simple liquid media, and it regularly produces amounts of proteolytic enzymes which are secreted into the culture medium.
As used herein, “meat” culture refers to a culture of cells grown in the presence of tissue derived from a mammal. “Meat” is not intended to include poultry or fish. As used herein, “meat-derived” and “animal-derived” are used interchangeably. Particularly, bovine products have been used in culture media in the fermentation of C. histolyticum, but they run the risk of contamination by agents which cause transmissible spongiform encephalopathies (TSEs; e.g., prions associated with bovine spongiform encephalopathy or “mad cow disease”). A known meat culture is the H4 strain of Clostridium histolyticum, which was developed in Dr. I. Mandl's laboratory at Columbia University in 1956. A further meat culture has been derived from the H4 strain named the ABC Clostridium histolyticum master cell bank, which has been deposited as ATCC 21000.
U.S. Pat. No. 7,811,560 (“the Auxilium '560 patent”), which is incorporated herein by reference in its entirety, discloses methods of producing collagenases without using meat-derived media. Using soybean derived fermentation medium, the methods described therein generated separately highly purified collagenase I and II, which were recombined in a 1:1 ratio to produce a drug product. The Auxilium '560 patent also discloses methods of producing highly purified collagenases using culture media containing porcine-derived products.
U.S. Patent Publication 2010/0086971 (“the Roche patent publication”), which is also incorporated herein by reference in its entirety, discloses numerous fermentation recipes which are based on vegetable peptone, including soybean-derived peptone, or vegetable-derived peptone plus fish gelatin. The Roche patent publication discloses successful growth of Clostridium and collagenase/gelatinase activities in these medium conditions, and does not disclose measuring clostripain. However, in another Roche patent U.S. Pat. No. 7,956,167, the inventors disclose that clostripain activity in the clostridia fermentation broth for downstream purification is “even more preferred between about 100 and 200 U per mg total collagenase”.
One drawback to known methods of fermenting Clostridium for the purposes of isolating collagenase is the production of contaminating proteases such as clostripain. Clostripain, a cysteine protease, is believed to be a major cause of collagenase degradation and instability. When proteases are present in a crude collagenase mixture, researchers must take extra precautions to neutralize the proteases, including making use of protease inhibitors, such as leupeptin, and performing all of the purification steps in specially designed cold rooms with chilled solutions. Theoretically, there are two approaches to dealing with the clostripain problem: one approach focuses on the downstream process, i.e., developing a purification method to remove clostripain as early as possible; the second approach is to develop a method to reduce clostripain production during the fermentation stage.
Thus, the art is in need of improved methods of culturing C. histolyticum. In view of this it is an objective of the invention to provide alternative methods and compositions for vegetable-derived media to support the growth of C. histolyticum and produce collagenase I and II in amounts which can be highly purified from culture supernatant in the absence of other contaminating proteases such as clostripain.
The inventors have surprisingly found that various components found in prior art fermentation media have a significant impact on the generation of clostripain, which leads to degradation of the desired collagenase I and II later in the purification process. Specifically, the inventors have found that either (1) minimizing the glucose content in the meat-free or vegetable-derived media; or (2) increasing the salt concentration in the meat-free or vegetable-derived media; or a combination of the two approaches, fulfills the above objective.